https://orcid.org/0000-0002-2902-3551
Figures
Abstract
Neisseria gonorrhoeae is classified by the Centers for Disease Control and Prevention as an urgent public health threat due to rising infections and rapid resistance development. N. gonorrhoeae has developed resistance to nearly all FDA-approved drugs, with ceftriaxone being the only remaining effective treatment for gonococcal infections. Alarmingly, ceftriaxone-resistant N. gonorrhoeae strains were isolated worldwide, raising the potential of untreatable gonorrhea in the near future. Hence, the critical need to develop new anti-N. gonorrhoeae therapeutics cannot be overemphasized. In this study, we identified the peptide mimetic brilacidin as an effective anti-gonococcal agent. Brilacidin completed phase 2 clinical trials for treating skin infections, oral mucositis, and COVID-19. Herein, brilacidin displayed potent activity against a panel of 22 drug-resistant strains of N. gonorrhoeae, inhibiting 50% of the strains tested (MIC50) at the concentration of 4 µg/mL. The peptide exhibited rapid bactericidal activity, reducing N. gonorrhoeae high inoculum within two hours. Moreover, brilacidin was superior to the drug of choice, ceftriaxone, in eliminating the intracellular N. gonorrhoeae harbored within endocervical cells. Additionally, brilacidin showed high tolerability in mammalian cells and lacked hemolytic activity in human erythrocytes. Altogether, the results demonstrate that brilacidin is a promising anti-gonococcal agent that warrants further in-depth investigation.
Citation: Abdelsattar AS, Abutaleb NS, Seleem MN (2025) A novel peptide mimetic, brilacidin, for combating multidrug-resistant Neisseria gonorrhoeae. PLoS One 20(6): e0325722. https://doi.org/10.1371/journal.pone.0325722
Editor: Ayesha Sabah Rahman, University of Birmingham School of Dentistry, UNITED KINGDOM OF GREAT BRITAIN AND NORTHERN IRELAND
Received: January 17, 2025; Accepted: May 16, 2025; Published: June 5, 2025
Copyright: © 2025 Abdelsattar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
Neisseria gonorrhoeae is the bacterium responsible for gonorrhea, one of the most prevalent sexually transmitted diseases [1]. In the United States, the Centers for Disease Control and Prevention (CDC) estimates that 1.6 million new gonococcal infections occur annually, which results in healthcare costs of about $135 million [2,3]. Globally, the World Health Organization (WHO) estimates indicate that over 82 million people were newly infected with gonorrhea in 2020 [4,5]. Given that many N. gonorrhoeae infections are asymptomatic, reported cases likely represent only a fraction of the true prevalence [6–11].
In addition to the high incidence rate of N. gonorrhoeae infections, the uprising antibiotic resistance rates in N. gonorrhoeae have become a serious public health concern. Hence, N. gonorrhoeae is classified by both the WHO and the CDC as a superbug and an urgent threat [12]. N. gonorrhoeae has developed resistance to nearly all FDA-approved therapies, including the last resort therapeutic for N. gonorrhoeae infections, ceftriaxone [13–16]. Worrisomely, N. gonorrhoeae resistance was extended to gepotidacin which is currently in clinical trials and has not been approved yet [17,18]. These rising resistance rates underscore the urgent need for novel anti-N. gonorrhoeae therapeutics.
Brilacidin is a synthetic peptide with demonstrated antifungal [19,20], antiviral [21–24], and antibacterial activity, particularly against the Staphylococcus aureus [25–27]. It has completed phase 2 clinical trials for treating S. aureus skin infection (NCT02052388), SARS-CoV-2 infections (NCT04784897), and as a rinse to treat oral mucositis (NCT02324335). However, brilacidin’s activity has not been evaluated against N. gonorrhoeae. Given the dearth of new anti-gonococcal therapeutics and the increased interest in repurposing brilacidin for treatment of microbial infections, the aim of this study is to investigate the anti-N. gonorrhoeae activity of brilacidin. We assessed the anti-gonococcal activity of brilacidin against multiple multidrug-resistant N. gonorrhoeae strains. Additionally, we examined its killing kinetics via a time-kill assay, cytotoxicity on endocervical cells, and hemolytic activity on the human red blood cells (RBCs). Brilacidin’s ability to clear intracellular N. gonorrhoeae within endocervical cells was also investigated. Finally, its mechanism of action was explored using ATP leakage and propidium iodide uptake assays.
2. Material and methods
2.1. Bacterial strains and reagents
N. gonorrhoeae strains were obtained from the CDC, the WHO, and the American Type Culture Collection (ATCC) (Table 1). The ME-180 cell line (ATCC HTB-33) was obtained from the ATCC. Antibiotics used in this work were purchased commercially: ciprofloxacin (Sigma-Aldrich, St. Louis, MO, USA), gentamicin (Chem-Impex International, Wood Dale, IL, USA), azithromycin, and ceftriaxone (TCI America, Portland, OR, USA), and brilacidin (MedChemExpress, Monmouth Junction, NJ, USA). Media and reagents including McCoy’s 5A medium and hematin (Sigma Aldrich, St. Louis, MO, USA), triton X-100 (Acros Organics, Fair Lawn, NJ, USA), BacTiter-Glo reagent (Promega Corporation, Madison, WI, USA), Propidium iodide (PI) and nicotinamide adenine dinucleotide (NAD) (Chem-Impex International, Wood Dale, IL, USA), MTS (3-(4,5-dimethylthia- zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Abcam, Waltham, MA, USA), and brucella broth, chocolate II agar plates, IsoVitaleX and bovine hemoglobin (Becton, Dickinson and Company, Cockeysville, MD, USA), were obtained from chemical vendors.
2.2. Antibacterial susceptibility analysis
The inhibitory activity of brilacidin and standard antibiotic drugs (ciprofloxacin, tetracycline, azithromycin, and ceftriaxone) was evaluated against 22 antibiotic-resistant N. gonorrhoeae strains using the broth microdilution method, as described elsewhere [28–31]. Briefly, N. gonorrhoeae colonies were collected and diluted in brucella supplemented broth to achieve a concentration of ~1 × 106 CFU/mL. Brilacidin and control antibiotics were then serially diluted in brucella supplemented broth across 96-well plates. Plates were incubated at 37 °C with 5% CO2 for 24 h to determine the minimum inhibitory concentrations (MICs).
2.3. Time-kill kinetics
The bactericidal activity of brilacidin against N. gonorrhoeae FA1090 was evaluated by assessing bacterial growth kinetics, as previously described [32,33]. Briefly, a logarithmic phase bacterial culture was diluted in the supplemented brucella broth to a final concentration of ~1 × 106 CFU/mL. Brilacidin and azithromycin were each added at 4 × MIC. Bacteria treated with dimethyl sulfoxide (DMSO) served as the negative control, while azithromycin served as a control antibiotic. Cultures were incubated with test agents at 37 °C for 24 h, with aliquots taken after 0, 2, 4, 6, 8, 10, 12, and 24 h, diluted and plated on chocolate II agar plates to determine the CFU.
2.4. Intracellular bacterial clearance assay
The intracellular bacterial clearance assay was performed to assess brilacidin’s ability to penetrate endocervical cells and eliminate the intracellular N. gonorrhoeae, as described elsewhere [29,34,35] with modifications. Briefly, the human endocervical epithelial cells (ME-180) were seeded into 96-well plates with McCoy’s 5A medium supplemented with 10% fetal bovine serum. ME-180 monolayers were then infected with N. gonorrhoeae FA1090 (multiplicity of infection (MOI) = 10) and incubated at 37°C with 5% CO2 for 24 h. Then, the phosphate-buffered saline (PBS) containing 320 μg/mL gentamicin was used to wash the wells three times before incubating with media containing gentamicin for one hour to kill the extracellular bacteria. Thereafter, PBS was utilized to wash the cells and they were subsequently treated with 4 × MIC of brilacidin, ceftriaxone, azithromycin, or DMSO (negative control). Plates were incubated at 37°C with 5% CO2 for 24 h. After incubation, the wells were washed with PBS and lysed with 2 mM EDTA and 0.5% saponin for one minute to release the intracellular bacteria for quantification.
2.5. Cytotoxicity and hemolysis assays
The potential toxic effect of brilacidin was evaluated using the ME-180 cell line, as described elsewhere [36–38]. Briefly, ME-180 cells were seeded and incubated with brilacidin at various concentrations (in triplicates) for 24 h. Cell viability was measured by monitoring the change of MTS color due to NADH reduction in viable cells, recorded at an absorbance of 490 nm (OD490).
Brilacidin’s hemolytic activity was evaluated following previously described methods [39,40]. Single-donor human RBCs (Innovative Research, MI, USA) were suspended in PBS at the concentration of 4% v/v. Brilacidin (in triplicate) was serially diluted in PBS to final concentrations of (16, 32, 64 and 128 μg/mL) and incubated with RBCs suspension at 37°C for one hour. Triton X-100 (0.1%) was used as a positive control to induce complete hemolysis, while PBS served as a negative control. After incubation, the erythrocytes were centrifuged at 800 × g for 10 min, and the absorbance of the supernatant was measured at 540 nm to assess hemolysis.
2.6. Permeability assays
Propidium iodide (PI) fluorescence assay was used to assess brilacidin’s ability to damage bacterial cytoplasmic membranes [41,42]. Briefly, N. gonorrhoeae (1 × 107 CFU/mL) was incubated with brilacidin (5× and 10 × MIC), azithromycin (10 × MIC), or triton X-100 (0.1%) in the presence of 10 μM PI for 1 hour. DMSO-treated N. gonorrhoeae served as a negative control. After incubation, the bacterial pellet was washed with PBS, and PI uptake was measured using a plate reader (excitation at 585 nm and emission at 620 nm).
In addition, an ATP leakage assay was used to assess the membrane integrity by measuring luminescence using the Luminescent ATP Detection Assay Kit according to the manufacturer’s instructions [43].
2.7. Statistical analyses
Each experiment was repeated at least twice. The GraphPad Prism 9.0 (Graph Pad Software, La Jolla, CA, USA) was used to generate the graphs and statistical analysis was conducted using one-way ANOVA (analysis of variance). Results were considered statistically significant if P-values < 0.05, and data are presented as means ± standard error of the mean.
3. Results and discussion
3.1. Anti-gonococcal activity of brilacidin
The anti-gonococcal activity of brilacidin was assessed against 22 multidrug-resistant N. gonorrhoeae isolates, including nine WHO reference strains with diverse resistance profiles and known phenotypic and genetic markers [44]. Brilacidin showed MIC values ranging from 1 to 8 µg/mL, inhibiting 90% of the strains (MIC90) at 8 µg/mL and 50% of strains (MIC50) at 4 µg/mL (Table 1). These strains showed high resistance levels to some control antibiotics. As illustrated in Table 1, ciprofloxacin had MIC50 and MIC90 values of 16 and >64 μg/mL, respectively, while tetracycline showed MIC50 of 2 and MIC90 of 8 μg/mL. Additionally, azithromycin displayed MIC50 of 1 and MIC90 of >64 μg/mL, and ceftriaxone presented MIC50 and MIC90 values of 0.032 and 1 μg/mL, respectively. These MICs for tetracycline, ciprofloxacin, azithromycin, and ceftriaxone align with previously reported values for these strains [44,45].
3.2. Killing kinetics of brilacidin
Brilacidin’s killing kinetics against N. gonorrhoeae WHO-X was evaluated in a time-kill assay. Remarkably, brilacidin (at 4 × MIC) demonstrated a rapid killing activity outperforming the control antibiotic azithromycin. As depicted in Fig 1, the burden of N. gonorrhoeae WHO-X was completely eradicated within 2 hours of treatment with brilacidin. However, azithromycin (at 4 × MIC) needed 6 hours to completely eradicate the N. gonorrhoeae burden. This rapid antibacterial activity of brilacidin is a highly desirable trait for treating N. gonorrhoeae, as it offers benefits such as limiting infection spread, reducing the likelihood of resistance development, shortening treatment duration and preventing disease progression which are key factors for controlling N. gonorrhoeae infections [46,47].
Each point is the mean of Log10 CFU/mL, and the error bars in each point are for the standard deviation of the mean.
3.3. Cytotoxicity and intracellular clearance activity of brilacidin
The ectocervical and endocervical cells in the female reproductive tract can be infected with N. gonorrhoeae, allowing the bacteria to survive intracellularly. N. gonorrhoeae has the ability to transmigrate across mucosal epithelial cells post invasion, potentially leading to disseminated infections. It can also inhibit the autophagy process during the invasion [48–50]. Most antibiotics are ineffective at reducing the burden of intracellular bacterial infections. For instance, ceftriaxone, the drug of choice, has limited activity against intracellular bacteria due to its high molecular weight (554.58 g/mol), low active transport, and high hydrophilicity (logP = 0.6) [51]. Given these limitations, we sought to evaluate the intracellular clearance activity of brilacidin using infected human endocervical epithelial cells (ME-180).
Initially, we tested the toxicity of brilacidin to endocervical epithelial cells, and found out it was well tolerated at a concentration up to 64 μg/ mL, with nearly 100% cell viability (Fig 2A). Hemolytic activity was also evaluated using human RBCs. Brilacidin showed an HC90 value (concentration causing 90% hemolysis) exceeding 128 μg/mL (Fig 2B), underscoring human cells’ tolerability to brilacidin. This broad therapeutic window, with minimal toxicity to mammalian cells and strong bactericidal activity, suggests brilacidin’s selectivity against N. gonorrhoeae.
(A) ME-180 cell viability of after incubation with different concentrations of brilacidin for 24 h. Results are shown as a percentage of cell viability relative to negative control (DMSO). (B) Hemolytic activity of brilacidin against human RBCs. The results are shown as percentage of RBCs hemolysis for each concentration of brilacidin relative to 0.1% Triton X-100 (positive control with complete hemolysis of RBCs). Error bars represent the standard deviation of the mean. **** (P < 0.0001), ns stands for not significant.
Subsequently, we evaluated brilacidin’s ability to clear the burden of intracellular N. gonorrhoeae within infected mammalian cells. As represented in Fig 3, brilacidin (at 4 × MIC) completely eradicated intracellular N. gonorrhoeae within 24 hours. Interestingly, brilacidin was superior to ceftriaxone, which showed a lower level of intracellular reduction for N. gonorrhoeae FA1090. These findings indicate that brilacidin can effectively penetrate host cells and eliminate N. gonorrhoeae at a rate superior to the drug of choice, ceftriaxone.
DMSO served as a negative control. Asterisks (*) denote statistically significant differences between test agents and DMSO (untreated) (P < 0.05). Pound signs (#) indicate statistically significant differences (P < 0.05) between brilacidin and azithromycin in comparison to ceftriaxone.
3.4. Mechanistic insights of brilacidin
The mechanism of action of drugs with rapid bactericidal activity, particularly antimicrobial peptides, is often mediated by disrupting the bacterial membrane [52]. Since brilacidin is a peptide mimetic with rapid bactericidal activity, we sought to investigate its membrane disruption activity against N. gonorrhoeae. Membrane disruption was evaluated by monitoring the fluorescence intensity of propidium iodide (PI) in N. gonorrhoeae FA1090. As illustrated in Fig 4A, the untreated bacteria or those treated with azithromycin had no significant difference in the fluorescence intensity, indicating no disruption of the cytoplasmic membrane integrity. In contrast, brilacidin’s treatment led to a significant fluorescence increase (intensity of ~1446 and 1787 at 5× and 10 × MIC, respectively), suggesting significant membrane disruption.
A) Propidium iodide fluorescence after treating N. gonorrhoeae with either azithromycin or brilacidin to predict the permeabilization of the cytoplasmic membrane. B) Percentage of ATP leakage from N. gonorrhoeae treated with brilacidin or azithromycin relative to Triton X-100 (positive control with complete ATP leakage). Asterisks denote statistically significant differences between test agents and DMSO (untreated), * (P < 0.05), ** (P < 0.01), and **** (P < 0.0001) as determined by one-way ANOVA.
Additionally, intact bacterial cells normally retain ATP, and extracellular ATP leakage indicates membrane disruption [53,54]. To verify the findings of the PI uptake assay, we measured the intracellular ATP level in N. gonorrhoeae cells after being treated with brilacidin compared to untreated bacteria. Triton X-100 (0.1%) was used as a positive control (considered as 100% ATP leakage). As demonstrated in Fig 4B, the supernatant of brilacidin-treated cells had a significant increase in the luminescent intensity compared to the untreated control, indicating a significant ATP leakage (~74% leakage).
These results align with previous reports demonstrating brilacidin’s membrane disruptive effect on various bacteria and fungi, including S. aureus, Aspergillus fumigatus, Cryptococcus gattii and C. neoformans [19,26,55].
4. Conclusion
This work demonstrated the anti-gonococcal activity of the peptide mimetic, brilacidin. Brilacidin displayed potent efficacy against multiple multidrug-resistant clinical isolates of N. gonorrhoeae with an MIC50 value of 4 µg/mL. In addition, brilacidin showed rapid bactericidal activity against the ceftriaxone-resistant strain WHO-X, outperforming azithromycin. It also outperformed ceftriaxone in clearing the burden of N. gonorrhoeae FA1090 inside infected mammalian cells. Mechanistically, brilacidin disrupted the gonococcal membrane, leading to ATP leakage and influx of propidium iodide inside the cells. These findings highlight the promising potential of brilacidin as a novel peptide mimetic to combat multidrug-resistant N. gonorrhoeae.
Acknowledgments
The authors would like to thank the CDC and the FDA Antibiotic Resistance Isolate Bank (Atlanta, GA) for supplying several of the clinical isolates used in this study.
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